Novel Tetraene Macrodiolides Are Effective Inducers of Mitochondrial Apoptosis in Jurkat Cells

Abstract

We synthesized 16 representatives of a new class of tetraene macrodiolides with two pharmacophore cis,cis-1,5-diene fragments of the molecule in their structure in rather high yields (from 67 to 84%), which, in turn, were synthesized by a catalytic intermolecular cyclocondensation reaction of α,ω-alka-nZ,(n+4)Z-diendiols with α,ω-alka-nZ,(n+4)Z-diendioic acids using Hf(OTf)₄. The synthesis of starting substrates with 1Z,5Z-diene moieties with a high degree of stereoselectivity was carried out using the authors' original reaction of catalytic homo-cyclomagnesiation of O-containing allenes. The cytotoxic potential of the examined compounds was assessed using the following cell lines: Jurkat, K562, U937, HL60, HEK293, and Wi-38 (fibroblasts). Biological tests of the synthesized compounds showed a direct effect on mitochondrial biogenesis by the dissociation of oxidation and phosphorylation and the release of cytochrome P450 into the cell cytosol, as well as the induction of mitochondrial apoptosis. The selectivity index demonstrates significant variability, ranging from approximately 2.5 to 5.3 for Jurkat cells and from 3.0 to 5.8 for the other cell lines.

Keywords: 1,5-dienoic compounds; antitumor agents; apoptosis; cyclocondensation; homo-cyclomagnesiation; macrodiolides; tetraenes.

Figure 1

Structures of some natural macrocycles.

Scheme 1

Synthesis of α,ω-alca-nZ,(n+4)Z-dienediols 4a–d and α,ω-alca-nZ,(n+4)Z-dienedioic acids 5a–d.

Scheme 2

Synthesis of tetraenoic macrodiolides 6–9a–d.

Figure 2

The cytotoxic activity (CC₅₀) of the synthesized tetraene macrodiolides was evaluated in vitro on cell cultures (Jurkat, K562, U937, HL60, HEK293, and fibroblasts). (A) Heat map of the values representing the level of cell toxicity. (B) Histogram of standard deviation values. The values presented in the figure correspond to the median for all cell lines, with individual values for each cell line provided in the heatmap. CC₅₀ was determined for all synthesized compounds. The incubation period was 24 h. The presence of three asterisks *** (p < 0.001) indicates the significance of the observed differences between the CC₅₀ values obtained in the samples treated with the synthesized compounds and the CC₅₀ values in fibroblast and the conditioned control cell line HEK293 (comparison was performed by one-way ANOVA). All experiments were performed in triplicate.

Figure 3

Detection changes in mitochondrial membrane potential (ΔΨ) and coupled early and late apoptosis in Jurkat cells treated with compounds 9d, 8d, 7d, and 6d. Staurosporine and carbonyl cyanide-m-chlorophenylhydrazone were utilized as control compounds. The addition of all the tested compounds was executed at the CC₅₀ concentration (refer to the provided table for the cytotoxicities of the synthesized compounds). The staining of cells was conducted using MitoSense Red, Annexin V-CF488A, and 7-AAD. The incubation period lasted for a duration of four hours. The presence of three *** (p < 0.001) and two asterisks ** (p < 0.005) indicates the significance of the observed differences between the values obtained in the control sample and the samples treated with the synthesized substances (comparison was performed by one-way ANOVA). All experiments were performed in triplicate.

Figure 4

Detection of cytochrome c in Jurkat cells stained with a FlowCellect Cytochrome c Kit. The control compound, carbonyl cyanide m-chlorophenylhydrazone (CCCP), was administered at a concentration equal to its CC50 value. Compound 9d was added to the sample cells at a CC50 concentration (see the table of cell viability). The cells were incubated for 6 h. We compared the numerical values to determine the reliability of the differences observed between the control sample and the samples treated with the synthesized substances. The presence of three asterisks *** (p < 0.001) indicates the significance of the observed differences between the values obtained in the control sample and the samples treated with the synthesized substances. We used a statistical t-test to perform this comparison. All experiments were performed in triplicate.